De Novo-designed Peptide Transforms Golgi-specific Lipids into Golgi-like Nanotubules

Abstract

Cellular organelles, such as the Golgi apparatus and the endoplasmic reticulum, adopt characteristic structures depending on their function. While the tubular shapes of these structures result from complex proteinlipid interactions that are not fully understood, some fundamental machinery must be required. We show here that a de novo-designed 18-mer amphipathic ␣-helical peptide, Hel 13-5, transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of and resembling the shape of the nanotubules that form the Golgi apparatus. Furthermore, we show that that the size and the shape of such nanotubules depend on lipid composition and peptide properties such as length and the ratio of hydrophobic to hydrophilic amino acids. Although the question of precisely how nature engineers organellar membranes remains unknown, our simple novel system provides a basic set of tools to begin addressing this question.Cellular organelles like the Golgi apparatus and the endoplasmic reticulum adopt characteristic structures depending on their function (1, 2). For example, extensive membrane nanotubules, typically 50 -70 nm in diameter and up to several m in length, have been observed to form the Golgi complex, the trans Golgi network, and the connections between the Golgi stacks. The morphological engineering of these membranes involve complex interactions between proteins and lipids that are not yet understood (3, 4). However, there must be some fundamental machinery required to form such structures.We recently described the properties of a de novo-designed 18-mer peptide, Hel 13-5 (5, 6). This peptide can adopt an ideal ␣-helix having a 240°hydrophobic sector region (Fig. 1A). It forms a self-association state in buffer solution by adopting this amphipathic structure (70% ␣-helical structure by CD), and it binds to model-and biomembranes with high affinity. In the present study, we show that Hel 13-5 induces nanotubular structures, not only for PC liposomes, but also for various naturally occurring phospholipids. Most importantly, we demonstrate that Hel 13-5 transforms spherical liposomes made from a Golgi-specific phospholipid mixture into nanotubules on the scale of, and resembling the shape of, the nanotubules of the Golgi apparatus. MATERIALS AND METHODSReagents-Peptide was synthesized by the Fmoc 1 strategy based on the solid phase technique starting from Fmoc-PAL-PEG resin using a PerSeptive 9050 automatic peptide synthesizer described previously (5). The stock solutions of Hel peptides were prepared as follows: the powders were damped with a small amount of 30% acidic acid and then diluted in buffer (5 mM Tes/100 mM NaCl, pH 7.4). The peptide concentrations in the buffer solution were determined from the UV absorbance of Trp at 280 nm (⑀ ϭ 5500).Turbidity Measurement-A lipid solution in chloroform was filmed in a round bottom flask by drying in a stream of N 2 gas. The lipid film was hydrated with the Tes buffer by vortexing. The turbid liposome solution obtained was then dilu...

A Hydrophobic Patch in a Charged α-Helix Is Sufficient to Target Proteins to Dense Core Secretory Granules

“…This finding made it possible to systematically characterize the biochemical requirements for this sorting activity. The initial peptide that we tested (K/LϩW) was shown previously to form an amphipathic helix and to rearrange Golgi-enriched liposomes into nanotubular structures in vitro (23). For direct comparison of the activity of this helix in in vitro liposome deformation and in vivo secretory granule sorting, we included the bulky hydrophobic tryptophan residue placed originally in this helix to measure tryptophan quenching in vitro (23).…”

“…The initial peptide that we tested (K/LϩW) was shown previously to form an amphipathic helix and to rearrange Golgi-enriched liposomes into nanotubular structures in vitro (23). For direct comparison of the activity of this helix in in vitro liposome deformation and in vivo secretory granule sorting, we included the bulky hydrophobic tryptophan residue placed originally in this helix to measure tryptophan quenching in vitro (23). Our results demonstrate that the presence of the hydrophobic leucine and tryptophan residues in the helix played a critical role in conferring sorting activity ( Fig.…”

“…Fusion proteins were constructed by selective amplification of the corresponding fragments using PCR. Attachment of the K/LϩW synthetic amphipathic ␣-helix (KLLKLLLKLWLKLLKLLL) (23) to the Fc fusion protein containing PC1/3-(711-740) (see Figs. 1 and 2) or the Fc protein alone (see Fig.…”

“…This G/LϩW helix-containing fusion protein showed no significant co-localization with POMC or ACTH in either the Golgi apparatus or the cyto- Isoelectric point and helical wheel projections were determined using the EMBOSS software package (27). Secondary structure was either predicted with the NNPREDICT (A) (24), GOR III (B) (25), and PROF (C) (26) algorithms or determined by circular dichroism (D) (23). Hydrophobic cluster analysis was carried out by the method of Gaboriaud et al (28).…”

Interfacial Behavior of Pulmonary Surfactant Preparations Containing Egg Yolk Lecithin

“…The Hel 13-5 NH 2 -KLLKLLLKLWLKLLKLLL-COOH peptide was synthesized using the 9-fluorenylmethoxycarbonyl Fmoc technique and purified using reverse-phase HPLC, as described in the literature 23 . More detailed procedures for the synthesis, purification, and analysis of Hel 13-5 were reported previously 24,25 . …”